Tomato plant resistant to tomato brown rugose fruit virus

ABSTRACT

The present invention relates to a tomato,  Solanum lycopersicum,  plant that is resistant to  Tobamovirus,  wherein the plant comprises one or more genomic sequences conferring  Tobamovirus  resistance. More specifically the invention relates to a tomato plant that is resistant to Tomato Brown Rugose Fruit Virus (TBRFV). The present invention further relates to a genomic sequence or locus providing resistance to  Tobamovirus.  In addition, the present invention relates to methods for proving a tomato plant that is resistant to  Tobamovirus.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of InternationalApplication No. PCT/EP2019/084272, filed Dec. 9, 2019, which claimspriority to International Application No. PCT/EP2019/050830, filed Jan.14, 2019, each of which is incorporated herein by reference in theirentirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name:701802018401SUBSEQLIST.TXT, date recorded: Apr. 14, 2021, size: 433 KB).

Description

The present invention relates to a plant of the S. lycopersicum speciesthat is resistant to Tobamovirus, wherein the plant comprises one ormore genomic sequences. More specifically the invention relates totomato plants (S. lycopersicum) that are resistant to Tomato BrownRugose Fruit Virus (TBRFV). The present invention further relates to agenomic sequence or locus providing resistance to Tobamovirus.Furthermore the present invention relates to methods for providing a S.lycopersicum plant that is resistant to Tobamovirus.

Tobamovirus is a genus in the virus family Virgaviridae that infectsplants, including plants of the Solanaceae family, such as tobacco,potato, tomato, and eggplant and are among the most serious threats tovegetable crops in the world. Tobamoviruses are particularly a problemin tomato crops grown in protected environments and are transmitted overlong distances through external seed contamination, and mechanicallyfrom plant to plant through common culture practices through workers'hands, clothes, tools, and are capable to preserve infectivity in seedsand contaminated soil. Furthermore, common weeds, often asymptomaticwhen infected by the virus comprise a cryptic reservoir between growthcycles.

Tobamovirus infections can have disastrous effects in crops when theybecome contaminated. Prevention of infection, by, for example, raisingseedlings in a virus free environment is generally costly and/orunfriendly to the environment. In addition, these methods do not alwaysprovide satisfactory results.

Tobamoviruses are non-enveloped, with helical rod geometries, andhelical symmetry. Viral particles are rod-shaped and have a diameter ofaround 18 nm, and a length of 300 to 310 nm. Their positive-sense singlestranded RNA genomes are linear and non-segmented, and around 6.3 to 6.5kb in length. There are over 35+ virus species in this genus includingTomato Mosaic Virus (ToMV) or Tobacco Mosaic Virus (TMV), Tomato MildMottle Virus (ToMMV), and the recently newly discovered Tomato BrownRugose Fruit Virus (TBRFV).

In tomato, naming of the four strains of Tobamovirus (more specificallyToMV) currently recognized (Tm-0, Tm-1, Tm-2 and Tm-2²) is based on theintrogressed resistance (R) genes Tm1, Tm2 and Tm2² from related wildspecies. The Tm1 gene was introgressed from Solanum habrochaites and isincompletely dominant. The Tm2 and Tm2² genes were introgressed fromSolanum peruvianum and confer dominant complete resistance to ToMV.However new strains of Tobamovirus have emerged as resistance isovercome and recently resistance-breaking Tobamovirus species have beenreported in commercial fields in Mexico, Jordan, and Israel.

In the end of 2014 and beginning of 2015 an outbreak of a new diseaseinfecting tomatoes occurred in Israel and Jordan. Symptomatic plantsshowed a mosaic pattern on leaves accompanied occasionally by narrowingof leaves and yellow spotted fruit. Research showed that this newdisease was a new Tobamovirus, called TBRFV. TBRFV infection isassociated with necrotic lesions on leaves and tomato plants show mildfoliar symptoms at the end of the season but strong brown rugosesymptoms on fruits, making the fruit unsuitable for consumption.Furthermore, regarding to other members of the Solanaceae family, itseems that the TBRFV is capable to infect pepper plants as well, e.g.when planted on contaminated soil from a previous growth cycle ofinfected tomato plants in high temperatures above 30° C.

In the battle against Tobamovirus, resistance was introduced in tomatoesby introgression of the R genes Tm2 and Tm2², resulting in resistance toToMV. However, these R-genes do not provide resistance to the new TBRFV,since different domains in the viral proteins comprised of differentprotein structure and a new resistance mechanism and/or resistant genesare required for a different resistance mechanism. Furthermore, it ishighly likely that over time resistance will be broken, since the viruswill adapt and evolve, resulting in viral breakthrough. Therefore, newresistance genes need to be identified and/or combined to provideresistant crops, especially against the new TBRFV.

Considering the above, there is a need in the art for TBRFV resistanttomato plants, more specifically TBRFV resistant S. lycopersicum. Inaddition, there is a need in the art to provide methods and means forproviding TBRFV resistant S. lycopersicum plants.

It is an object of the present invention, amongst other objects, toaddress the above need in the art. The object of present invention,amongst other objects, is met by the present invention as outlined inthe appended claims.

Specifically, the above object, amongst other objects, is met, accordingto a first aspect, by the present invention by a plant of the S.lycopersicum species that is resistant to Tobamovirus, wherein the plantcomprises a TBRFV resistance gene that encodes for a TBRFV resistanceprotein, wherein the protein has at least 90%, preferably at least 95%,more preferably at least 98%, even more preferably at least 99%, mostpreferably 100% amino acid sequence identity with SEQ ID No.116. It ispredicted that the TBRFV resistance gene encodes for a NBS-LRRresistance protein.

According to a preferred embodiment, the present invention relates tothe plant, wherein the TBRFV resistance gene comprises a coding sequencethat has at least 90%, preferably at least 95%, more preferably at least98%, even more preferably at least 99%, most preferably 100% nucleotidesequence identity with SEQ ID No.115.

According to another preferred embodiment, the present invention relatesto the plant, wherein the plant comprises one or more genomic sequencesselected from the group consisting of SEQ ID No.1, SEQ ID No.2 SEQ IDNo.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8,SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQID No.14, SEQ ID No.15, SEQ ID No.16, SEQ ID No.17 and SEQ ID No.18, orhaving at least 95% sequence identity with any of said SEQ ID No's. Thegenomic sequences encode for one or more genes or genetic elements thatprovide resistance to Tobamovirus. Sequences have been examined on genehomology using public database of the National Center for BiotechnologyInformation (NCBI). Six genomic sequences have homology with sequencesthat encode for NBS-LRR resistance proteins (SEQ ID No.7, 8, 9, 10, 11and 14). Four genomic sequences have homology with LRR receptor-likeserine/threonine-protein kinase (SEQ ID No. 5, 6, 12 and 13).

Pathogen recognition by plants takes place via two relevant groups ofhost receptors involving two main types of proteins, namelyReceptor-like kinases or proteins (RLK or RLP) and nucleotide-bindingsite leucine-rich repeat proteins (NBS-LRR resistance proteins). Thefirst group are pattern recognition receptors (PRR) specializing in therecognition of pathogen associated molecular patterns (PAMPS). RLPs orRLKs are attached to the cell membrane and are extracellular immunereceptors. Plant RLKs are involved in plant-pathogen interaction anddefence responses and plant receptor kinases (PRKs) can be defined asproteins that contain an extracellular domain, a single-passtransmembrane domain and a cytoplasmic serine/threonine (Ser/Thr)protein kinase domain. Plant LRR-RLKs (leucine rich-repeat receptor-likekinase) possess a functional cytoplasmic kinase domain, and all of theplant LRR-RLKs analysed to date possess Ser/Thr kinase activity. Theresistance to pathogens provided by these receptors is calledPAMP-triggered immunity (PTI). The other group mainly comprisesintracellular receptors called resistance proteins (R proteins). Themajority of disease resistance genes in plants encode nucleotide-bindingsite leucine-rich repeat proteins, also known as NBS-LRR proteins. Theseproteins are characterized by nucleotide-binding site (NBS) andleucine-rich repeat (LRR) domains as well as variable amino- andcarboxy-terminal domains and are involved in the detection of diversepathogens, including bacteria, viruses, fungi, nematodes, insects andoomycetes. The majority of the identified genomic sequences that provideTobamovirus resistance comprise multiple LRR domains. It is thought thatthese domains determine effector recognition and therefore diseasesusceptibility/resistance.

Pathogens develop counter strategies to overcome PTI through modifyingor changing PAMPs or MAMPs. Then, plants will develop a way to recognizethese effectors and trigger a faster and stronger secondary defenceresponse known as effector-triggered immunity (ETI). ETI is mediated byR proteins and accompanied by localized cell death around the site ofinfection. The presence of these newly identified resistance gene and/orgenomic regions encoding NBS-LRR proteins and/or plant receptor kinaseswill decrease the chances of the pathogen overcoming the resistance, orwhen combined with other resistance genes, disease resistance may evenbe further improved.

According to a preferred embodiment, the present invention relates tothe plant, wherein the plant comprises the genomic sequence representedby SEQ ID No.3. The genomic sequence SEQ ID No. 3 comprises multiplesequences that have homology with sequences that encode for NBS-LRRresistance proteins and LRR receptor-like serine/threonine-proteinkinase.

According to yet another preferred embodiment, the present inventionrelates to the plant, wherein the plant comprises SEQ ID No.8, SEQ IDNo.9, SEQ ID No.10 and SEQ ID No.11.

According to the present invention, Tobamovirus resistance of the plantmay be affected by one or more genomic sequences encoding a NBS-LRRprotein selected from the group of SEQ ID No.8, No.9, No.10, No.11 andNo.14, for example a combination of SEQ ID No.8 and SEQ ID No. 9, or SEQID No.8 and SEQ ID No.10, SEQ ID No.8 and SEQ ID No. 11, SEQ ID No. 9and SEQ ID No. 10, SEQ ID No.9 and SEQ ID No.11, SEQ ID No.10 and SEQ IDNo. 11. Furthermore or alternatively, the resistance may affected by oneor more genomic sequences encoding a LRR receptor-likeserine/threonine-protein kinase selected from the group of SEQ ID No.12, SEQ ID No.13, or SEQ ID No.12 and SEQ ID No.13.

According to yet another preferred embodiment, the present inventionrelates to the plant, wherein the plant comprises the genomic sequencesof SEQ ID No.8, SEQ ID No.11 and SEQ ID No.14.

According to a preferred embodiment, the present invention relates tothe plant, wherein the plant comprises SEQ ID No.8, SEQ ID No.9, SEQ IDNo.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13 and SEQ ID No.14.

According to another preferred embodiment, the present invention relatesto the plant, wherein the plant is resistant to Tobamovirus strainsTm-0, Tm-1 and Tm-2. In tomato, four strains of Tobamovirus have beenidentified; Tm-0, Tm-1, Tm-2 and Tm-2².

According to yet another preferred embodiment, the present inventionrelates to the plant, wherein the plant is resistant to Tomato BrownRugose Fruit Virus (TBRFV).

According to yet another preferred embodiment, the present inventionrelates to the plant, wherein the TBRFV is virus isolate AE050.

According to yet another preferred embodiment, the present inventionrelates to the plant, wherein the plant is a tomato plant (Solanumlycopersicum).

According to yet another preferred embodiment, the present inventionrelates to the plant, wherein the one or more genomic sequences and/orTBRFV resistance gene is heterozygously or homozygously present in thegenome of said plant. From the experimental data it can be concludedthat the resistance is dominant and that the TBRFV resistance geneand/or genomic sequence must be at least heterozygously present in thegenome of the plant to provide resistance against the Tobamovirus.

According to yet another preferred embodiment, the present inventionrelates to the plant, wherein said TBRFV resistance gene and/or one ormore genomic sequences are as found in the deposit accession numberNCIMB 43279. At least 625 seeds of tomato (Solanum lycopersicum) variety‘2018.11310 T 15233-11’ were deposited on Nov. 16, 2018 according to theBudapest Treaty in the National Collection of Industrial, Food andMarine Bacteria Ltd (NCIMB Ltd), Ferguson Building, Craibstone Estate,Bucksburn, Aberdeen, AB21 9YA, United Kingdom. The deposit has beenassigned NCIMB number 43279. Viability testing of the deposit wascompleted. Access to this deposit will be available during the pendencyof this application to persons determined by the Commissioner of Patentsand Trademarks to be entitled thereto under 37 C.F.R. § 1.14 and 35U.S.C. § 122. Upon allowance of any claims in this application, allrestrictions imposed by the depositor on the availability to the publicof the deposited material will be irrevocably removed for theenforceable life of the patent. The deposit will be maintained in theNCIMB depository, which is a public depository, for a period of at least30 years, or at least 5 years after the most recent request for a sampleof the deposit, or for the effective life of the patent, whichever islonger, and will be replaced if the deposit becomes nonviable duringthat period.

The present invention, according to a second aspect, relates to plants,plant parts, tissues, cells, and/or seeds derived from the plant of thepresent invention.

The present invention, according to a further aspect, relates to aresistance gene (TBRFV resistance gene) for providing resistance to aTobamovirus in a S. lycopersicum plant, wherein said resistance gene isrepresented by a coding sequence having at least 90% nucleotide sequenceidentity with SEQ ID No.115.

The present invention, according to a further aspect, relates to agenomic sequence for providing resistance to a Tobamovirus in a S.lycopersicum plant, wherein the genomic sequence is selected from thegroup consisting of SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8,SEQ ID No.9, SEQ ID No.10, SEQ ID No.11, SEQ ID No.12, SEQ ID No.13, SEQID No.14, SEQ ID No.15, SEQ ID No.16, SEQ ID No.17 and SEQ ID No.18, orhaving at least 95% sequence identity with any of said SEQ ID No's.Preferably the genomic sequence is SEQ ID No.8, SEQ ID No.11 or SEQ IDNo.14.

The present invention, according to a further aspect, relates to aresistance locus for providing resistance to a Tobamovirus in a S.lycopersicum plant, wherein the locus is represented by SEQ ID No.1, SEQID No.2, SEQ ID No.3 or SEQ ID No.4, preferably SEQ ID No.3.

According to a preferred embodiment of present invention, the resistancegene, genomic sequence or resistance locus provides resistance to aTBRFV.

The present invention, according to a further aspect, relates to amethod for providing a plant of the S. lycopersicum species that isresistant to Tobamovirus, wherein the method comprises the steps of;

-   -   a) selecting a S. habrochaites plant that is resistant to        Tobamovirus, wherein said selection comprises establishing the        presence of the resistance gene genomic sequence or resistance        locus of present invention,    -   b) transferring the identified genomic sequence or locus of        step a) into a S. lycopersicum plant thereby conferring        Tobamovirus resistance to said S. lycopersicum plant.        Transferring can be done by crossing the selected S.        habrochaites plant with a S. lycopersicum. Subsequently, a        Tobamovirus resistant S. lycopersicum plant can be selected.

According to another preferred embodiment, the present invention relatesto the method, wherein after step b) a first S. lycopersicum plant isselected that is resistant to Tobamovirus and is crossed with a secondS. lycopersicum plant that is not resistant to Tobamovirus, andsubsequently selecting S. lycopersicum plants that are resistant toTobamovirus.

According to a preferred embodiment, the present invention relates tothe method, wherein in step a) establishing the presence of theresistance gene (TBRFV resistance gene), resistance conferring genomicsequence or the resistance locus in a S. habrochaites plant is performedby one or more markers selected from the group consisting of SEQ ID No:83, SEQ ID No: 84, SEQ ID No: 85, SEQ ID No: 86, SEQ ID No: 87, SEQ IDNo: 88, SEQ ID No: 89, SEQ ID No: 90, SEQ ID No: 91, SEQ ID No: 92, SEQID No: 93, and SEQ ID No: 94, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No.107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 110, SEQ ID No. 111, andSEQ ID No. 112, preferably SEQ ID No. 105, SEQ ID No. 106, SEQ ID No.107, SEQ ID No. 108, SEQ ID No. 109, SEQ ID No. 110, SEQ ID No. 111, andSEQ ID No. 112.

The present invention, according to a further aspect, relates to a useof a marker for establishing the presence of the TBRFV resistance gene,the TBRFV resistance conferring genomic sequence or the resistance locusin a S. lycopersicum plant, wherein the marker is one or more markersselected from the group consisting of SEQ ID No: 83, SEQ ID No: 84, SEQID No: 85, SEQ ID No: 86, SEQ ID No: 87, SEQ ID No: 88, SEQ ID No: 89,SEQ ID No: 90, SEQ ID No: 91, SEQ ID No: 92, SEQ ID No: 93, and SEQ IDNo: 94, SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No. 108,SEQ ID No. 109, SEQ ID No. 110, SEQ ID No. 111, and SEQ ID No. 112,preferably SEQ ID No. 105, SEQ ID No. 106, SEQ ID No. 107, SEQ ID No.108, SEQ ID No. 109, SEQ ID No. 110, SEQ ID No. 111, and SEQ ID No. 112.

The present invention will be further detailed in the following examplesand figures wherein:

FIG. 1: Shows an overview of the mapping of the locus providingresistance against Tobamovirus, more specifically TRBFV in S.lycopersicum. F1 plants were created by crossing S. habrochaites line90479-3 that was selected for resistance phenotype with S. lycopersicumlines OT9 and OT1317 to create two populations for mapping. Over 700plants were tested on TRBFV resistance and several recombinant plants(21 plants) were selected and the results of the disease test werecombined with the marker data. Several molecular markers (M1 to M42)have been used to determine the position and size of the genomicsequence providing resistance to TRBFV. A clear segregation was observedbetween resistant (R) and susceptible plants (S). The results indicatedthat a genomic region located between markers M16 and M17 was providingthe TRBFV resistance; the corresponding locus (named LYC4943 R Locus) is133.515 bp in length and comprises several putative genes. Based onfurther fine mapping, the size and location of the genomic sequence thatwas harbouring the TBRFV resistance was determined to be between markersM33 and M38 and was approximately 68.000 bp larger compared to theSL2.40 reference genome of S. lycopersicum (85.240 bp vs. 17.328 bp,respectively). It is therefore most likely that one or more genes thatare located within this region, indicated as “TBRFV region”, areproviding the TBRFV resistance.

FIG. 2: Shows the results of a qPCR for the detection of TBRFV ininfected and uninfected tomato plants (S. lycopersicum) of the presentinvention and plants that do not comprise the TBRFV resistance locus.Low Ct values (i.e. below 30) indicate the high presence of viral RNApresent in the samples. OT9 is a tomato plant that does not comprise theTBRFV resistance locus. The control sample (OT9 uninfected) showed a Ctvalue of between 35 and 40 cycles and the infected control sample (OT9infected) showed a Ct value of between 20 and 25. Plants that show a Ctvalue above 30 cycles, preferably around 35 cycles were consideredresistant, whereas plants that show a Ct value below 30 were consideredsusceptible. Tomato plants comprising the TBRFV resistance locus,homozygous (B) or heterozygous (H) have a Ct value above 30 cycles andwere considered as being resistant. Furthermore, the results showed thatthe resistance is dominant Plants that did not comprise the TBRFVresistance locus (A and OT9 infected) showed a Ct value of between 20and 25, indicating that the plant was susceptible to TBRFV infection.

FIG. 3: Shows a schematic overview of the genomic sequences SEQ ID No.1to SEQ ID No.18 of the present invention that encode for one or moregenes or genetic elements that provide resistance to Tobamovirus.

FIG. 4: Shows an overview of a further mapping of the locus providingresistance against Tobamovirus in addition to FIG. 1. A furtherrecombinant selection has been performed by further genotyping plantswith M33 and M38 to identify recombinant plants in the identified TBRFVregion and further limit this TBRFV region. Recombinant plants have beenfurther genotyped with markers (M-SEQ 10, M-SEQ 11-1, M-SEQ 11-2, andM-SEQ 14) covering the TBRFV locus and were specifically designed toeliminate candidate regions in the TBRFV locus, i.e. the genomicsequences SEQ ID No.1 to SEQ ID No.18 of present invention that encodefor one or more genes or genetic elements that provide resistance toTobamovirus. Recombinant plants 15321-02, 15321-03 and 15321-07 werescreened for resistance by inoculation with TBRFV isolate AE50. Based onthese recombinant plants in combination with phenotyping, ELISA and qPCRdata for the determination of TBRFV infection, it was concluded that thegene conferring resistance is part of genomic SEQ ID No 14. Plants15321-02 and 15321-03 do not comprise SEQ ID No 14 and were shown to besusceptible to TBRFV, with high ELISA scores and low qPCR ct-values thatcorrespond to the values obtained with the susceptible control line OT9,indicating virus infection. Plant 15321-07 comprises the SEQ ID No 14and was shown to be resistant to TBRFV, with low ELISA scores and highqPCR ct-values.

FIG. 5: Shows the TBRFV infection by ELISA in a homozygous TBRFVresistant line (15322-04) as well as a susceptible control line (OT9).Plants were infected with TBRFV (+TRBFV) and infiltrated with constructVIGS-01a that specifically targets the TRBFV resistance gene or withconstruct VIGS-01b which targets a different region within theidentified TRBFV region. ELISA reading was done by measurement ofabsorption at 405 nm. Control plants OT9 infected by TBRFV resulted inabsorption levels of 2000 abs or higher, whereas the resistant plantlines infected with TRBFV resulted in absorption levels of approximately500 abs. In cases where the TRBFV resistance gene was silenced byVIGS-01a in the resistant plant lines, absorption levels of between 1500and 2250 abs were measured, indicating viral infection. Silencing byVIGS-01b in the resistant plant lines, resulted in similar absorptionlevels as was observed in the infected resistant plant lines that werenot silenced by VIGS.

FIG. 6: Shows the TBRFV infection by qPCR in a homozygous TBRFVresistant line (15322-04) as well as a susceptible control line (OT9).Plants were infected with TBRFV (+TRBFV) and infiltrated with constructVIGS-01a that specifically targets the TRBFV resistance gene or withconstruct VIGS-01b which targets a different region within theidentified TRBFV region. The infected control sample showed a Ct valueof approximately 12 or 13. The resistant plant lines infected with TRBFVshowed a Ct value of approximately 30, indicating TBRFV resistance. Incases where the TRBFV resistance gene was silenced by VIGS-01a in theresistant plant lines, Ct values were observed to drop to betweenapproximately 12 to 20, higher than the infected control cells, andclearly indicating viral infection. Silencing by VIGS-01b in theresistant plant lines, resulted in similar Ct levels (Ct value of ˜30)as was observed in the infected resistant plant lines that were notsilenced by VIGS.

EXAMPLES

Inoculation of a Tomato Plant with TBRFV

The TBRFV isolate AE050 (Origin: Saudi Arabia) was used to perform thedisease assays. As plant material, the Line OT9, which is a plant linesusceptible for TBRFV, was used for virus maintenance. Symptomaticleaves received from the original samples were used for sap-mechanicalinoculation on the Line OT9. The virus was maintained on systemicallyinfected tomato plants OT9 by monthly sap-mechanical inoculation on new3 weeks-old seedlings.

The tomato plants of the species of Solanum pennellii, S. peruvianum, S.chilense, S. habrochaites, S. pimpinellifolium, S. neorickii, S.corneliomulleri, S. chmielewskii, S. cheesmaniae, S. galapagense havebeen screened (˜800 out of 912 wild Solanum accessions in total). Twelveplants of each accession were infected with TBRFV isolate AE050.

Seeds were sown in vermiculite, seedlings were transplanted in rockwoolblocks and inoculated at 4 weeks after sowing. As starting materialsymptomatic leaves from infected-OT9 were collected and ground in amortar and pestle in chilled demi water with carborundum (1 gr/100 mL).The oldest leaf from 3 weeks-old seedlings of each test plant wasmechanically inoculated with AE050 by gently rubbing the leaf once withone finger.

The plants were phenotyped by visual scoring of the plants and theleaves. Plants were scored for visual symptoms at regular timeintervals. Symptoms were visually assessed at 2, 4 and 6 weeks afterinoculation, and ELISA tests on remaining plants were done from 6 weeksonwards, with 1 month intervals. More than 50% of the plants showedalready symptoms at 2 weeks after inoculation.

Visual scoring was performed on a weekly basis. Plants were scored forvisual symptoms. The presence of yellowing, mosaic pattern on leaves,leaf deformation (narrowing, mottling) was recorded on a weekly basis atthe plant level. First symptoms were typically observed 12-14 dayspost-inoculation. Plants were categorized as “resistant” when no suchsymptoms on leaves were observed. Plants displaying any of the symptomson leaves were categorized as “susceptible”. Leaf samples were collectedfrom asymptomatic plants (i.e. resistant) to test for the presence ofvirus by ELISA.

The screening allowed the selection of several candidates for resistancebreeding, with the best candidate being LYC4943, a S. habrochaitesaccession from Peru. LYC4943 was symptomless and tested virus-free byELISA for more than 15 weeks after inoculation.

Determination of TBRFV Infection by ELISA

Infection was determined by ELISA. One apical leaf (fully expanded) ofevery plant was collected. Leaves were crushed using a Type R302D63N-472 machine (VECTOR Aandrijftechniek B.V., Rotterdam, TheNetherlands) and sap was collected by adding 2 mL of PBS-Tween buffer.100 μL of the extract was used for ELISA with antibodies against ToMV(supplier Prime Diagnostics, Wageningen, The Netherlands). ELISA readingwas done by measurement of absorption at 405 nm with a FLUOstar Galaxyapparatus. Plants that gave absorption values more than 1.5 times of theclean control plants were considered infected (susceptible).

Bioassays and Mapping of TBRFV Resistance Genomic Sequence

The original LYC4943 (S. habrochaites) seed lot was segregating for theresistance. Nine different F1 families were sown for bioassay in orderto identify the F1 families which were completely resistant (resistanceis fixed) and which would be used for further backcrosses. Four F1families germinated and were tested in bioassay. F1 plants created withLYC4943 plant 3 (90479-3) were selected for resistance phenotype tocreate two populations for mapping, choosing the S. lycopersicum linesOT9 and OT1317 as backcross lines. Markers M1 to M42 (respectively SEQID No. 19 to SEQ ID No.102) that have been used in the mapping arelisted in Table 1.

298 plants (OT9×90479−3)×OT9) and 484 plants (OT1317×90479−3)=total of782 plants were inoculated with the TBRVF isolate AE050. Two to threeweeks after inoculation the TBRFV symptoms were present and phenotypingby eye was done. A clear segregation was observed between resistant (R)and susceptible plants (S) and resistant phenotypes could be linked withmarker M1 (see Table 1) located on chromosome 8 at 2673609 bp on thereference genome SL2.40 (S. lycopersicum). 92 plants have been genotypedwith 26 markers in order to flank the QTL (M2 to M27, see Table 1).Based on these results the resistance could be mapped between 53118984bp and 57038544 bp on the reference genome SL2.40 (between M8 & M20, SeeTable 1 and FIG. 1).

TABLE 1 Pos. Pos Primer name Primer sequence SL2.40 on Locus SEQ ID No.M1_F GGTACAACAATTGACCAAGG  2672994  19 M1_R GCTAATTAAAAAGGAACATCAGC  20M2_F GCTATGGCGGAGAAGTCAAG    18124  21 M2_R AGTCACCTCCATAGTAGACC  22M3_F GGATCCAAGTTGTGTTCGAAC   881036  23 M3_R CTTCTCATCAATGTATGTGATTTC 24 M4_F TGTATAACACCTGGTGCTCC 15384575  25 M4_R CCATTTTCTGTTACAAAATTTCAG 26 M5_F GCTTCCCAATTTATGCTGAAG 47887679  27 M5_R GAGCCTCCCACTATAGTAATC 28 M6_F AGAATTATCATTTGCAGGATCG 50957946  29 M6_R CTATGGTTCGCATGTCATGC 30 M7_F CACAACGGCAATATACCTTGC 53082561  31 M7_R TGGAAGTATTAGAAAGGTCCAG 32 M8_F CCATTGAGAATAACTACTGTAC 53118984  33 M8_R CCACAGGATGACTAACTTGG 34 M9_F TGCAGTATTGATCGCATCTTCTA 53452252  35 M9_R GTTTGTTGCTGCCCTCAAA 36 M10_F TGATCAAGAATTTTGTTTTAGCATAGA 55664335  37 M10_RTAAAGCATCAATTTTGCATTGTCT  38 M11_F TCGAAGACTAACAAAGTCCTTGTAGA 55720872 39 M11_R GACACTCCGGCAGTTCCTT  40 M12_F TTCTTATGTGAAAAATTGGGTGG 55776574 41 M12_R ACTACGCAGTCCCACAGCTT  42 M13_F TTGTTTGGTGGATCCATGTG 56448988 43 M13_R AGGGAAAGGGCAAGGATG  44 M14_F GATCTACCAATGGCTATTCATC 56781521 45 M14_R GCAAAACTTAACCGGTCTAAG  46 M15_F TCTCGATGGTTGATAATTTGTTC56874054  47 M15_R GGAATCGATTAACACTGGTTC  48 M16_F CATCTTATTGAAGCTCTGCTG56920720  49 M16_R CAAACAGTCCCTATTCAACAC  50 M17_F GGTCTTGCGCTAATCAAAAG56990004  51 M17_R GCGTTGTGGTGAAAGTTTTATC  52 M18_F CTTGTTTGGATGGTTGTCAC57003163  53 M18_R CAACAAAAAATATACAATCCGTCC  54 M19_FGAGATAGAAGGAAACTTACCG 57024614  55 M19_R CAATTATCCCCTCAGTTCTG  56 M20_FTATGCCTGTCCCTGAAAAGG 57038544  57 M20_R AGGGTCTTGGATCAAATCTTGA  58 M21_FTGTGGACTTGGAGTGGTATC 57427631  59 M21_R GTAGAAAGGGTAGGCATGTTC  60 M22_FTACCAAAGCAAACACTGCCAC 57441418  61 M22_R AGCCACGAGATATATATTGGAG  62M23_F GATAAGACCGCCAATAACTAG 60844273  63 M23_R GTGATCTCCATGAGCAAATG  64M24_F TGAGTTGAGATGCTGTTCTAG 61412883  65 M24_R AGTCCACCAAGACTTAAAGAG  66M25_F GTCTGCCTTCTCTTGCATGC 62277547  67 M25_R GTTGCTCCAGACAGAATAAGC  68M26_F CATCGAAGAGATGTGTAGGG 62418391  69 M26_R TGCAGTTGAAGTAGACTTCAG  70M27_F TCAACGTTAGTGGTGATGCTAG 62783214  71 M27_R CAATTGCAGAAAGTGAAGCTG 72 M28_F GTGGATTCAGTTAAACCAGAAC 56924513   4076  73 M28_RGACATGTGGAACTTGACAAAAC  74 M29_F GCGAGAGAAAAGATTCTCTAC 56934501  12765 75 M29_R CATTCTTCACTCTCTCAAGATG  76 M30_F CGTTTGGTGATCTGCCTTGTCTT56934846  13109  77 M30_R TCTTCTTGTAGGGAATCCAGAATC  78 M31_FGTGTCCTGTGCTTGTTATTCC 56935054  13317  79 M31_R CCTCAAACCTATTGCATCTGACA 80 M32_F CGGCTCAGCGAGGAAGTGCAG 56935849  14113  81 M32_RCGTTGACTGTTTTTCTTTATG  82 M33_F GTAAGCTCCTTCATGTCAGC 56941043  15893  83M33_R CAAGTATTGTCTGCCGAGTAAC  84 M34_F GCGTACAGACATATTTATGCAAC 56942927 17777  85 M34_R GAACAGCTAAAAGTAAGAGCAC  86 M35_F GTTCATGTGTGTTTATGGACC56943610  18416  87 M35_R CTTCACTAAATAAATAAGTGGTAG  88 M36_FTATGGATTTGTGTCTCAGAAGA 56944105  18912  89 M36_R TGTGGTCACCAAGTGGGTTTC 90 M37_F GTCTTCCAGAGCAGTTATGCAAG 56945167  19974  91 M37_RTGAGACTGCTAAGTTGACTTGTTTG  92 M38_F GTACACCAAATCACAGACATCG 56958371101133  93 M38_R CCCAATTTGGTTTGTGTTGGAC  94 M39_F GAAATTCCTTGCCTCCTCTC56961307 104063  95 M39_R GTGGAAGCCATAGTGTACAAG  96 M40_FCATATTATACAGTGAAAGCTTTG 56965103 107926  97 M40_R GAATTGCAGTTCACTTGCTTC 98 M41_F CCACAAAGCTAAAAAGGGATTG 56969685 112529  99 M41_RTCCATGTGAGTTTTGTGTGTG 100 M42_F GCCACATAAATTACATATAGCTG 56981278 125792101 M42_R GAACTATTCAACAAGCATAATAC 102 M-SEQ 10_FGTCTTACAATAGTAAAATGCGCAG  36480 105 M-SEQ 10_R GCGGTTCGTTGATATTCCAAC 106M-SEQ 11_1F AGCGAAAGCGGAAGGAGTAC  48748 107 M-SEQ 11_1RTGTGGTGAGTAAGCAATGAATC 108 M-SEQ 11_2F GTGTATAATTCGCCAGAATATACGG  52303109 M-SEQ 11_2R CGTTTAGATAATTGTATATTACACATATG 110 M-SEQ 14FCAAATTATTACTTATGTTGTGATTTG  77410 111 M-SEQ 14R ATTAAGCCATGATACACAAATTAC112

The whole population of 782 plants have been genotyped with the flankingmarkers M8 & M20 in order to find the recombinant plants for furtherfine mapping. This resulted in 21 recombinant plants (See FIG. 1). These21 recombinant plants have been selected and genotyped with 11 markersM9 to M19 in order to further fine map the region (Table 1). Theresistance could be fine mapped between 56920720 and 56990004 (markerM16 and M17) on the reference genome SL2.40.

Sequencing the resistant LYC4943 region using Oxford Nanopore sequencingtechnology resulted in a locus of 133.515 bp. The 21 recombinant plantshave been genotyped with extra markers in this specific locus (M28 toM42) of LYC4943. Based on the recombinant plants, plants 594 and 608, itwas determined that the resistant region was located between positions56941043 and 56958371, based on the reference genome SL2.40,corresponding with positions between 15.893 and 101.133 on the LYC4943locus (between M33 and M38, see FIG. 1).

Based on the fine mapping, the size and location of the genomic sequencethat was harbouring the TBRFV resistance was determined to be betweenmarkers M33 and M38 and was approximately 68.000 bp larger compared tothe SL2.40 reference genome of S. lycopersicum (85.240 bp vs. 17.328 bp,respectively). It is therefore highly likely that one or more genes arelocated within this region, indicated in FIG. 1 as “TBRFV region”,providing the TBRFV resistance and is indicated as SEQ ID No.3 in thisapplication. Based on the reference genome SL2.40 and in silicoprediction analysis (ITAG 2.3), at least one gene is located in the finemapped region that encodes for a CC-NBS-LRR resistance protein. Blastingthe fine mapped TBRFV region against the database of National Center forBiotechnology Information (NCBI), resulted in seven genomic fragments ofwhich five have homology with NBS-LRR resistance proteins (SEQ ID No.8,No.9, No.10, No.11 and No.14) and two have homology with LRRreceptor-like serine/threonine-protein kinases (SEQ ID No.12 and SEQ IDNo.13).

Next, further fine mapping was performed and a recombinant selection hasbeen performed by genotyping 668 BC2 plants ((OT9×90479−3)×OT9×OT9) withM33 and M38 in order to identify recombinant plants in the TBRFV region,which resulted in three plants 15321-02, 15321-03 and 15321-07 (see FIG.4). These three plants were tested for resistance by inoculation withTBRFV isolate AE50. Approximately three weeks after TBRFV inoculationthe plants were phenotyped by observation, and ELISA and qPCR wasperformed to monitor virus infection. The recombinant plants have beengenotyped with markers (M-SEQ 10, M-SEQ 11-1, M-SEQ 11-2, and M-SEQ 14,respectively SEQ ID No. 105 to SEQ ID No. 112) covering the TBRFV locusand were specifically designed to eliminate candidate genes in the TBRFVlocus. This approach provided insight into which of the candidategenomic sequences SEQ ID No.1 to SEQ ID No.18 of present inventionspecifically provides resistance to TBRFV. Based on the recombinantplants and phenotyping by disease tests, ELISA and qPCR, we concludedthat the gene conferring resistance is encoded by genomic sequence ofSEQ ID No 14, more specifically the coding DNA sequence of SEQ ID No.115 encoding the protein of SEQ ID No. 116.

Validation Tm0, Tm1 & Tm2 Strain Resistance in Plant Comprising theTBRFV Resistance Locus

A tomato plant of the present invention (S. lycopersicum) comprising theTBRFV resistance locus (SEQ ID No. 1) was tested for resistance againstthe Tm0, 1 and 2 strains. The presence of the TBRFV resistance locus wasdetermined by markers M16, M17 and M33. It was furthermore confirmedthat the plant does not contain the Tm2² gene (is a known gene thatprovides resistance against Tm0, 1 and 2 strains). In some case theplant did contain the Tm1 resistance gene. As a control, plants wereselected that did not contain the TBRFV resistance locus.

Eight plants (See Table 2, 1 to 8) of which six plants comprise theTBRFV resistance locus (heterozygous), and two plants (7 and 8) do nothave the TBRFV resistance locus have been inoculated with the Tm0isolate. Eight plants (See Table 2, 9 to 16) of which six plantscomprise the TBRFV resistance locus (heterozygous), and two plants (15and 16) do not have the TBRFV resistance locus, have been inoculatedwith the Tm-1 isolate. Eight plants (See Table 2, 17 to 28) of whichfour plants comprise the TBRFV resistance locus (two homozygous 17, 18+two heterozygous 19, 20), and four plants not have the TBRFV resistancelocus have been inoculated with the Tm2 isolate. As control thesusceptible cultivated tomato line OT95 was also inoculated with allthree strains.

First symptoms were typically observed after 12-14 dayspost-inoculation. Plants were categorized as Resistant (R) when nomosaic pattern symptoms on leaves were observed; plants displaying anyof the symptoms on leaves were categorized as Susceptible (S). Thephenotype of every single plant has been compared with the TBRFVgenotype. Results are summarized in Table 2 below.

TABLE 2 Plant Isolate Phenotype M16 M33 M17 Tm2 Tm1  1 Tm0 R h h h a a 2 Tm0 R h h h a a  3 Tm0 R h h h a a  4 Tm0 R h h h a h  5 Tm0 R h h ha h  6 Tm0 R h h h a h  7 Tm0 R a a a a h  8 Tm0 R a a a a h OT95 Tm0 Sa a a a a OT95 Tm0 S a a a a a  9 Tm1 R h h h a a 10 Tm1 R h h h a a 11Tm1 R h h h a a 12 Tm1 R h h h a a 13 Tm1 R h h h a h 14 Tm1 R h h h a h15 Tm1 S a a a a a 16 Tm1 S a a a a h OT95 Tm1 S a a a a a OT95 Tm1 S aa a a a 17 Tm2 R b b b a a 18 Tm2 R b b b a a 19 Tm2 R h h h a a 20 Tm2R h h h a a 21 Tm2 S a a a a a 22 Tm2 S a a a a a 23 Tm2 S a a a a a 24Tm2 S a a a a a OT95 Tm2 S a a a a a OT95 Tm2 S a a a a a R = resistant,S = susceptible, a = no resistance locus present, h = heterozygous, b =homozygous

Result Tm0

All plants that contained the TBRFV resistance locus were resistant.Plants 7 and 8 did not contain the TBRFV resistance locus but were alsoresistant. A reason that could explain the results is that the Tm1 geneis causing the resistance to ToMV isolate Tm-0. In addition, plant 1, 2and 3, did not contain the Tm1 gene, but did contain the TBRFVresistance locus, showed to be resistant.

Result Tm1

The resistant phenotypes are linked with the TBRFV genotypes, providingresistance against ToMV isolate Tm-1.

Result Tm2

The resistant phenotypes (hetero-, homozygous) are linked with the TBRFVgenotypes, providing resistance against ToMV isolate Tm-2.

Determination of TBRFV Infection in Tomato (S. lycopersicum) by qPCR

Tomato plants comprising the TBRFV resistance locus (heterozygous orhomozygous) and plants not containing this region have been selected forTBRFV bioassay using markers (M16 and M17). Plants were infected withTBRFV and the susceptible tomato line OT9 has been included as control(OT9 non-infected and infected).

After 3 weeks of inoculation, one leaf from the top of the plant ofevery single plant was collected in a 2 ml tube which contain a 6.35 mmmetal bullet. The tube was frozen in liquid nitrogen. The tubes wereshaken with high speed to pulverize the plant material. After spin downthe tube, the standard RNA extraction using Macherey-Nagel™ NucleoSpin™RNA Plant was carried out. RNA concentration was measured usingDropSense 96 (Trinean) and was diluted to a concentration of 100 ng/μl.900 ng have been used for cDNA synthesis using M-MLV ReverseTranscriptase (Invitrogen). 10 ng cDNA was used for Real-time PCR usingLC green as Intercalating dye. Two primer combinations for amplifyingthe TBRFV strain were used, see Table 3 (SEQ ID No.103 and SEQ IDNo.104, respectively).

TABLE 3 qPCR primer name Sequence TBRFV-3 Fw ACCGTTCAACGGCAATTTAGCTBRFV-3 Rev CCTATACACCTTAAAACCACTG

The more viral RNA present in the samples the lower the Ct value in theqPCR, since less PCR cycles are required to amplify the cDNA (of theviral RNA) and pick up a signal. The control sample (OT9 uninfected)showed a Ct value of between 35 and 40 cycles and the infected controlsample (OT9 infected) showed a Ct value between 20 and 25. Thereforeplants that show a Ct value above 30 cycles, preferably around 35 cycleswere considered resistant, whereas plants that show a Ct value below 30were considered susceptible (see FIG. 2).

Tomato plants comprising the TBRFV resistance locus, homozygous (B) orheterozygous (H) all have a Ct value above 30 cycles and can beconsidered as resistant. The results are showing that the resistance isdominant. Plants that did not comprise the TBRFV resistance locus (A)showed a Ct value of between 20 and 25, indicating that the plant wassusceptible to TBRFV infection.

Sequencing of Genomic Sequence of Resistant Tomato Plant

Genomic DNA was isolated from a resistant plant (S. lycopersicum) of thepresent invention, i.e. comprising the TBRFV resistance locus, accordingto the protocol as published on 27 Apr. 2018 in Nature, ProtocolExchange (2018), Rachael Workman et al. “High Molecular Weight DNAExtraction from Recalcitrant Plant Species for Third GenerationSequencing”. The sequencing libraries were prepared using the PCR free,no multiplex, DNA Ligation Sequencing Kit-Promethion (SQK-LSK109). Theisolation procedure resulted in high quality sequencing libraries to beused in the Oxford Nanopore system for sequencing (ONT sequencing).Promethion Flowcell Packs (3000 pore/flowcell) version R9.4.1. were usedfor sequencing.

Furthermore, to further resolve the TBRFV locus and identify the geneproviding the TBRFV resistance, we performed ONT sequencing on aresistant line (LYC4943). Sequencing of the entire transcript isoformsof the resistant LYC4943 line was done using the Iso-Seq analysisapplication (Pacific Biosciences of California, PacBio). This resultedin only one candidate resistance transcript/gene located in regionbetween markers M33 and M38, more specifically the TBRFV resistance geneof SEQ ID No. 115. This transcript was predicted to encode for aCC-NBS-LRR resistant protein of SEQ ID No. 116.

Gene Validation using VIGS

To confirm that the TBRFV resistance gene (SEQ ID No 115) was indeed thegene conferring resistance to TBRFV, a Virus Induced Gene Silencing(VIGS) analysis was performed. Tobacco rattle virus (TRV)-derived VIGSvectors have been abundantly described to study gene function in plantssuch as Arabidopsis thaliana, Nicotiana benthamiana, Lycopersiconesculentum and other plants (see for example Huang C, Qian Y, Li Z, ZhouX.: Virus-induced gene silencing and its application in plant functionalgenomics. Sci China Life Sci. 2012; 55(2):99-108).

As such, two VIGS constructs were developed (Table 4), one constructVIGS-01a to specifically target SEQ ID No 115 and a control constructVIGS-01b that targets SEQ ID No. 7, i.e. a sequence also located withinthe previously identified TBRFV locus.

TABLE 4 VIGS construct Sequence VIGS-01aGGAAGATTTTAATGAAAAGAGGTTGATAAAGA (SEQ ID No. 113)AAATTGTAGAATCTATTGAAGAAAAGTCACTT GGTGACATGGACTTGGCTCCACTTCAAAAGAAGCTTCAGGACTTGCTGAATGGAAAAAAATATT TGCTTGTCTTAGATGATGTTTGGAATGAAGATCAAGATAAGTGGGCTAAGTTAAGACAAGTCTT GAAGGCTGGAGCAAGTGGTGCTTATGTTCTAACCACTACC VIGS-01b AGAAGATTTTGATGAGAAGAAGTTGATAAAGG (SEQ ID No. 114)CAATTGTTGAATCTATCGAAGGAAACCCACTT GGTGACCACATGGATTTGGCTCCACTTCAAAAGAAGCTTCAGGACATGTTGAATGGAAAGAGAT ACTTTCTCGTTTTGGATGATGTTTGGAATGAAAATCAAGAAAAGTGGGATAAGATAAAAGCAGT CTTAGAGGTTGGAGCACGAGGTGCTTCTGTTCTAACCACCACT

The VIGS fragments were synthesized (IDT, gBlocks) and subsequentlycloned into a TRV vector. The DNA sequences were confirmed by Sangersequencing. The vector contained all sequences encoding for proteinsthat are required for a functional TRV particles including the targetsequences. The VIGS vectors including the VIGS-01a and VIGS-01bconstructs were transformed into Agrobacterium tumefaciens strain GV3101and used in VIGS experiments to reduce endogenous mRNA levels in tomatoplants used in this experiment. A homozygous TBRFV resistant line(15322-04) as well as a susceptible control line (OT9) were used in theVIGS experiment, in which plants were Agrobacterium infiltrated atseedling stage (cotyledons) followed by TBRFV isolate E50 inoculationthree weeks after Agrobacterium infiltration. Two weeks after TBRFVinoculation the individual plants were phenotyped by ELISA and qPCR andthis revealed that susceptibility was found in resistant plantsinfiltrated with construct VIGS-01a whereas no susceptibility had beendetected in resistant plants infiltrated using construct VIGS-01b.Results of the ELISA and qPCR are shown in FIGS. 5 and 6, and resultshave been summarized in Table 5.

TABLE 5 VIGS- TBRFV # S # R Plants # plants construct infection plantsplants R line 15322-04  7 VIGS-01a Yes 7  0 R line 15322-04  6 VIGS-01bYes 0  6 R line 15322-04 10 No Yes 0 10 S line OT9  6 No Yes 6  0

In the OT9 line all plants were susceptible, as expected. The R linewhich was shown earlier to be fully resistant became susceptible toTBRFV in cases where the suspected TBRFV resistance gene was silencedusing the VIGS-01a construct designed to specifically target this gene,whereas silencing using the VIGS-01b construct (control construct) didnot result in any susceptibility of the plants tested. Based on theseresults it can be concluded that gene SEQ ID No 115 is the conferringresistance to TBRFV.

1. A Tobamovirus resistant Solanum lycopersicum plant, wherein the plant comprises a Tomato Brown Rugose Fruit Virus (TBRFV) resistance gene encoding a TBRFV resistance protein comprising polypeptide sequence SEQ ID NO:
 116. 2. The plant of claim 1, wherein the resistance gene comprises nucleotide sequence SEQ ID NO:
 115. 3. The plant of claim 1, wherein the plant is resistant to Tobamovirus strains Tm0, Tm1, and Tm2.
 4. The plant of claim 3, wherein the plant is resistant to Tobamovirus strain Tm0.
 5. The plant of claim 3, wherein the plant is resistant to Tobamovirus strain Tm1.
 6. The plant of claim 3, wherein the plant is resistant to Tobamovirus strain Tm2.
 7. The plant of claim 1, wherein the plant is resistant to Tomato Brown Rugose Fruit Virus (TBRFV).
 8. The plant of claim 2, wherein the resistance gene is as found in the deposit under NCIMB Accession Number
 43279. 9. A tissue, cell, or plant part of the plant of claim
 1. 10. A seed of the plant of claim
 1. 